Epidemiological and clinical studies have found that cholesterol gallstones are more prevalent in women than in men at all ages in every population that has been studied. Accumulated evidence clearly shows that the use of oral contraceptive steroids and conjugated estrogens in women significantly increases the prevalence of cholesterol gallstones. Estrogen therapy to men with prostatic cancer also leads to similar lithogenic effects. These findings show that the increased risk of gallstones in women compared to men is related to differences in how the liver metabolizes cholesterol in response to estrogen. Our published studies have established a central role for estrogen by activating hepatic estrogen receptor ? (ER?), but not ER?, in promoting gallstone formation. However, the mechanisms mediating estrogen?s lithogenic actions on gallstone formation have become more complicated with the identification of a novel estrogen receptor, the G protein-coupled receptor 30 (GPR30). Furthermore, Gpr30 has been mapped to mouse chromosome 5 and is co-localized with Lith18, a new gallstone gene. Our molecular and genetic data support the candidacy of the Gpr30 gene as a compelling gene underlying Lith18. However, identifying the lithogenic mechanism of GPR30 remains a significant challenge because it is not yet fully understood whether GPR30 plays a major role in estrogen-induced gallstones and whether it acts independently of or in conjunction with ER? on inducing cholesterol gallstones. Our central hypothesis is that GPR30 is also involved in estrogen-dependent lithogenic actions, working independently of ER?, as both GPR30 and ER? can work through different pathways to promote the formation of estrogen-induced gallstones. The rationale for the proposed research is that once the particular mechanisms as to how estrogen increases susceptibility to gallstone formation through GPR30 are understood, the key components of estrogen signaling could be manipulated pharmacologically, leading to innovative targets to the treatment of gallstones in women. Guided by our preliminary data, we propose to test this hypothesis by pursuing three specific aims: first, to investigate the phenotypic characterization of GPR30 that determines susceptibility to cholesterol cholelithiasis; second, to study whether GPR30 activation of EGFR leads to the lithogenesis of bile by inhibiting hepatic bile acid synthesis in response to high levels of estrogen; and third, to elucidate the critical role of GPR30 in hepatic hypersecretion of biliary cholesterol and gallbladder hypomotility that accounts for rapid growth of cholesterol crystals. This application is innovative because distinguishing the lithogenic actions of GPR30 from those of ER? and further investigating how estrogen produces lithogenic actions through GPR30 will help elucidate all the molecular mechanisms behind the formation of estrogeninduced cholesterol gallstones. The proposed research is significant because it translates basic research discovery to a pre-clinical model designed to identify novel treatment targets and may provide an efficacious novel strategy for the prevention of gallstones in women and in patients exposed to high levels of estrogen.